Our broad, long-term goal is to define the molecular mechanisms by which the human DNA mismatch repair (MR) pathway processes DNA adducts and directs repair to one strand. When certain damaged DNA bases, such as those that result from chemotherapy with cisplatin or alkylating agents, are processed as mismatches, they can induce apoptosis. Clinically significant drug resistance is conferred when MR is silenced. We will define the molecular mechanism by which two of the most effective anticancer drugs, cisplatin and adriamycin, interact with the mismatch repair pathway. In addition, we have developed a new, straightforward method for constructing plasmids that contain a mismatch and a single nick in either DNA strand. This method will be extended to replace mismatches with one of several DNA modifications. Using this approach, we will characterize the mechanism by which covalent DNA damage is recognized and processed by MR. DNA modifications will include cisplatin, transplatin, O6- methylguanine, and 8-oxoguanine. An essential aspect of defining how DNA damage is processed is understanding how strands are distinguished. We will test several possible models by which MR can be targeted to one DNA strand. First, hemimethylation will be tested by constructing mismatch-containing molecules where one strand is selectively methylated at CpG-containing sequences by annealing fully-methylated homoduplex molecules with unmethylated, single-stranded partners. Assays will be performed in vitro, using nuclear extracts from MR-competent HeLa cells. Both covalently closed relaxed and supercoiled molecules will be tested. If hemimethylation is competent to direct MR, we will define the minimal sequence requirements for strand discrimination. We will also ask whether strand gaps or DNA ends can target MR. Free DNA ends, in particular the DNA end in the active site of replicative polymerases, have been proposed to target MR. We will test this model by asking whether free DNA ends, or defined gaps within circular DNAs, are capable of directing MR to the discontinuous strand.